3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-alkylamides as a new class of synthetic histone deacetylase inhibitors. 1. Design,synthesis, biological evaluation,and binding mode studies performed through three different docking procedures

Recently we reported a novel series of hydroxamates, called 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides (APHAs), acting as HDAC inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069-2072). Among them, 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide 1 was chosen as lead compound, and its binding mode into the modeled HDAC1 catalytic core together with its histone hyperacetylation, antiproliferative, and cytodifferentiating properties in cell-based assays were investigated (Mai, A.; et al. J. Med. Chem. 2002, 45, 1778-1784). Here we report the results of some chemical manipulations performed on (i) the aroyl portion at the C4-pyrrole position, (ii) the N(1)-pyrrole substituent, and (iii) the hydroxamate moiety of 1 to determine structure-activity relationships and to improve enzyme inhibitory activity of APHAs. In the 1 structure, pyrrole N(1)-substitution with groups larger than methyl gave a reduction in HDAC inhibiting activity, and replacement of hydroxamate function with various non-hydroxamate, metal ion-complexing groups yielded poorly active or totally inactive compounds. On the contrary, proper substitution at the C4-position favorably affected enzyme inhibiting potency, leading to 8 (IC50 = 0.1 micro M) and 9 (IC50 = 1.0 micro M) which were 38- and 3.8-fold more potent than 1 in in vitro anti-HD2 assay. Against mouse HDAC1, 8 showed an IC50 = 0.5 micro M (IC50 of 1 = 4.9 micro M), and also in cell-based assay, 8 was endowed with higher histone hyperacetylating activity than 1, although it was less potent than TSA and SAHA. Such enhancement of inhibitory activity can be explained by the higher flexibility of the pyrrole C4-substituent of 8 which accounts for a considerably better fitting into the HDAC1 pocket and a more favorable enthalpy ligand receptor energy compared to 1. The enhanced fit allows a closer positioning of 8 hydroxamate moiety to the zinc ion. These findings were supported by extensive docking studies (SAD, DOCK, and Autodock) performed on both APHAs and reference drugs (TSA and SAHA).     

3-(4-Aroyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides as a new class of synthetic histone deacetylase inhibitors. 3. Discovery of novel lead compounds through structure-based drug design and docking studies

Aroyl-pyrrole-hydroxy-amides (APHAs) are a new class of synthetic HDAC inhibitors recently described by us. Through three different docking procedures we designed, synthesized, and tested two new isomers of APHA lead compound 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1), compounds 3 and 4, characterized by different insertions of benzoyl and propenoylhydroxamate groups onto the pyrrole ring. Biological activities of 3 and 4 were predicted by computational tools up to 617-fold more potent than that of 1 against HDAC1; thus, 3 and 4 were synthesized and tested against both mouse HDAC1 and maize HD2 enzymes. Predictions of biological affinities (K(i) values) of 3 and 4, performed by a VALIDATE model (applied on either SAD or automatic DOCK or Autodock results) and by the Autodock internal scoring function, were in good agreement with experimental activities. Ligand/receptor positive interactions made by 3 and 4 into the catalytic pocket, in addition to those showed by 1, could at least in part account for their higher HDAC1 inhibitory activities. In particular, in mouse HDAC1 inhibitory assay 3 and 4 were 19- and 6-times more potent than 1, respectively, and 3 and 4 antimaize HD2 activities were 16- and 76-times higher than that of 1, 4 being as potent as SAHA in this assay. Compound 4, tested as antiproliferative and cytodifferentiating agent on MEL cells, showed dose-dependent growth inhibition and hemoglobin accumulation effects.     

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Sch?chtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.     

辛二酰苯胺异羟肟酸对人卵巢癌细胞恶性表型的抑制作用

目的 探讨辛二酰苯胺异羟肟酸(SAHA)对人卵巢癌细胞恶性表型的作用及其可能的分子作用机制。方法 （1）选取 2 组人卵巢癌细胞(SKOV3 和 SKOV3/DDP; HO8910 和 HO8910-PM)后, 设置对照组和终浓度为 1、 3、 5 及 7 μmol/L SAHA 组(SAHA 1~4 组), 采用 CCK-8 法分别检测 SAHA 对细胞增殖的影响。（2） 设置对照组和 SAHA 1、 2 组(2 和 5 μmol/L), Annexin V-FITC/PI 双标记流式细胞术分别检测 SAHA 对细胞凋亡及周期的影响。（3） RT-PCR 和 Western blot 检测 SAHA 1~4 组表型相关蛋白的表达水平。结果 （1）随着 SAHA 浓度的增加, SKOV3 细胞株 SKOV3/DDP 及 HO8910 细胞株 48 h 光密度 （OD） 值均呈逐渐降低趋势(均 P＜0.05)。（2） 48 h SAHA 1、 SAHA 2 组凋亡率高于对照组(均 P＜0.05); SAHA 作用 48 h, 细胞周期发生阻滞, 与对照组比较, SAHA 1 组和 SAHA 2 组 SKOV3 和 SKOV3/DDP 细胞 S 期和 G2/M 期比例增高, HO8910 和 HO8910-PM 细胞 G0/G1期比例增高(P＜0.05)。（3） SAHA 1、 2 组 CyclinB1 和 Cdc2(p34)的 mRNA 表达水平均低于对照组, Caspase-3、 p21 和 p53 的 mRNA 表达水平明显高于对照组(P < 0.05); SAHA 1~4 组 Ac-Histone H3 和 Ac-Histone H4 和 p53 蛋白的表达较对照组明显增强, CyclinB1、 Cdc2 (p34)蛋白的表达减弱。结论 SAHA 通过调节恶性表型相关蛋白 Caspase-3、 p53、 CyclinB1 和 Cdc2(p34)的表达水平, 促进组蛋白乙酰化, 抑制卵巢癌细胞增殖, 阻滞细胞周期并诱导细胞凋亡。     

II. Synthesis and biological evaluation of some bioisosteres and congeners of the antitumor agent, 2-(4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy)propionic acid (XK469)

XK469 (1) is among the most highly and broadly active antitumor agents to have been evaluated in our laboratories. Subsequent developmental studies led to the entry of (R)-(+) 1 (NSC 698215) into phase 1 clinical trials (NIH UO1-CA62487). The antitumor mechanism of action of 1 remains to be elucidated, which has prompted a sustained effort to elaborate a pharmacophoric pattern of 1. The present study focused on a strategy of synthesis and biological evaluation of topologically based, bioisosteric replacements of the quinoxaline moiety in the lead compound (1) by quinazoline (4a-d), 1,2,4-benzotriazine (12a-18b), and quinoline (21a-g) ring systems. The synthetic approach to each of the bioisosteres of 1 utilized the methodology developed in previous work (see Hazeldine, S. T.; Polin, L.; Kushner, J.; Paluch, J.; White, K.; Edelstein, M.; Palomino, E.; Corbett, T. H.; Horwitz, J. P. Design, Synthesis, and Biological Evaluation of Analogues of the Antitumor Agent 2-(4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy)propionic acid (XK469). J. Med. Chem. 2001, 44, 1758-1776.), which is extended to the procurement of the benzoxazole (23a,b), benzthiazole (23c,d), pyridine (25a,b), and pyrazine (27) congeners of 1. Only quinoline analogues, bearing a 7-halo (21a,b,d,e) or a 7-methoxy substituent (21g), showed antitumor activities (Br > Cl > CH(3)O > F approximately I), at levels comparable to or greater than the range of activities manifested by 1 and corresponding analogues. At high individual dosages, the (S)-(-) enantiomers of 1 and 21b,d all produce a reversible slowing of nerve-conduction velocity in the mice, the onset of which is characterized by a distinctive dysfunction of the hind legs, causing uncoordinated movements. The condition resolves within 5-10 min. However, at higher dosages, which approach a lethal level, the behavior extended to the front legs, lasting from 20 min to 1 h. By contrast, the (R)-(+) forms of these same agents did not induce the phenomenon of slowing of nerve-conduction velocity.     

Tricyclic pyrazoles. 3. Synthesis, biological evaluation, and molecular modeling of analogues of the cannabinoid antagonist 8-chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide

A series of analogues of 8-chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide 4a (NESS 0327) (Ruiu, S.; Pinna, G. A.; Marchese, G.; Mussinu, J. M.; Saba, P.; Tambaro, S.; Casti, P.; Vargiu, R.; Pani, L. Synthesis and Characterization of NESS 0327: A Novel Putative Antagonist of CB1 Cannabinoid Receptor. J. Pharmacol. Exp. Ther. 2003, 306, 363-370) was synthesized and evaluated for their affinity to cannabinoid receptors. Depending on the chemical modification of the lead structure that was chosen, compounds 4b, 4c, 4i, 4l, and 4m still proved to be potent binders of the CB1 receptor. Moreover, several analogues (4c, 4d, 4e, and 4m) demonstrated superior CB2 receptor binding affinities compared to the parent ligand. Compounds 4b, 4c, 4i, and 4l displayed the most promising pharmacological profiles, having the highest selectivity for CB1 receptors with Ki(CB2) to Ki(CB1) ratios of 11,250, 2000, 3330 and 4625, respectively. Compound 4c increased the intestinal propulsion in mice and antagonized the effect induced by the CB1 receptor agonist WIN 55,212-2. Finally, molecular modeling studies were carried out on a set of tricyclic pyrazoles (2a-4a) and on rimonabant 1 (SR141716A), indicating that high CB1 receptors affinities were consistent for the tricyclic derivatives, both with a nonplanar geometry of the tricyclic cores and with a precise orientation of the substituent (chlorine) on this ring system.     

Study on selectin blocker. 8. Lead discovery of a non-sugar antagonist using a 3D-pharmacophore model

We have developed a pharmacophore model of a ligand/E-selectin complex to screen drug candidates for selectin blockers. In a series of sugar mimetic studies of the E-selectin ligand, sialyl Lewis X (sLe(x)), we have already found a potent compound, a sulfated Le(x) analogue (1), and also have proposed how compound 1 binds to E-selectin (Tsujishita, H.; Hiramatsu, Y.; Kondo, N.; Ohmoto, H.; Kondo, H.; Kiso, M.; Hasegawa, A. J. Med. Chem. 1997, 40, 362-369). To find drug candidates that fit into the binding pocket of E-selectin, we constructed an original 3D-pharmacophore model from structural information of a compound 1/E-selectin complex model and screened lead compounds for selectin blockers using a commercially available database ACD-3D. As a result, we discovered a lead compound (2) containing good selectin inhibitory activity, and in addition, we succeeded to preliminarily optimize it to a more active lead compound (3) with micromolar IC(50) values, based on the 3D-pharmacophore model investigation. This methodology using the 3D-pharmacophore model could be applicable as a pre-screen system for selectin blockers.     

Exploiting subsite S1 of trypsin-like serine proteases for selectivity: potent and selective inhibitors of urokinase-type plasminogen activator.

A nonselective inhibitor of trypsin-like serine proteases, 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine (1) (Verner, E.; Katz, B. A.; Spencer, J.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.; Luong, C.; Martelli, A.; Radika. K.; Rai, R.; She, M.; Shrader, W.; Sprengeler, P. A.; Trapp, S.; Wang, J.; Young, W. B.; Mackman, R. L. J. Med. Chem. 2001, 44, 2753-2771) has been optimized through minor structural changes on the S1 binding group to afford remarkably selective and potent inhibitors of urokinase-type plasminogen activator (uPA). The trypsin-like serine proteases(1) that comprise drug targets can be broadly categorized into two subfamilies, those with Ser190 and those with Ala190. A single-atom modification, for example, replacement of hydrogen for chlorine at the 6-position of the 5-amidinoindole P1 group on 1, generated up to 6700-fold selectivity toward the Ser190 enzymes and against the Ala190 enzymes. The larger chlorine atom displaces a water molecule (H(2)O1(S1)) that binds near residue 190 in all the complexes of 1, and related inhibitors, in uPA, thrombin, and trypsin. The water molecule, H(2)O1(S1), in both the Ser190 or Ala190 enzymes, hydrogen bonds with the amidine N1 nitrogen of the inhibitor. When it is displaced, a reduction in affinity toward the Ala190 enzymes is observed due to the amidine N1 nitrogen of the bound inhibitor being deprived of a key hydrogen-bonding partner. In the Ser190 enzymes the affinity is maintained since the serine hydroxyl oxygen O gamma(Ser190) compensates for the displaced water molecule. High-resolution crystallography provided evidence for the displacement of the water molecule and validated the design rationale. In summation, a novel and powerful method for engineering selectivity toward Ser190 proteases and against Ala190 proteases without substantially increasing molecular weight is described.     

Northern Ring Conformation of Methanocarba-Adenosine 5'-Triphosphate Required for Activation of P2X Receptors

Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IVReplacement of the ribose moiety of adenosine 5'-triphosphate (ATP) with a carbocyclic ring constrained in either the Northern (N) or Southern (S) conformation produces agonists with widely differing activities at P2Y receptors (Kim et al. [2002] J Med Chem 45:208-218). We have used whole cell patch clamp recording to investigate the agonist activity of these two methanocarba analogs of ATP at four different P2X receptors (P2X(1), P2X(2), P2X(3), and P2X(2/3)). On dorsal root ganglion neurons, (N) methanocarba-ATP ((1'S,2'R,3'S,4'R,5'S)-4-(6-amino-9H-purin-9-yl)-1-[triphosphoryloxymethyl] bicyclo[ 3.1.0]hexane-2,3-diol; MRS 2340) activated rapidly-desensitizing (P2X(3)) and slowly-desensitizing (P2X(2/3)) receptors with a similar potency to ATP. In contrast, (S) methanocarba-ATP ((±)-5-(6-amino-9H-purin-9-yl)-1-[triphosphoryloxymethyl] bicycle [3.1.0]hexane-2,3-diol MRS 2312) was devoid of agonist activity. On nodose ganglion neurones, that express mainly P2X(2/3) receptors, ATP evoked a slowly desensitizing inward current with an EC(50) value of 26 μM. MRS 2340 was an effective agonist, but less potent than ATP, while MRS 2312 at concentrations up to 100 μM produced a barely detectable response. On mammalian cell lines expressing recombinant hP2X(1) and hP2X(2) receptors, MRS 2340 evoked inward currents similar in amplitude to those produced by the same concentration of ATP or α,β-mATP. In contrast, MRS 2312 failed to give a detectable response. Although the conformation of the ribose affects agonist activity at P2Y receptors, there is a strong requirement for the (N) conformation for the activation of these P2X receptors. Furthermore, the region of the agonist binding site that accommodates the ribose moiety appears to be highly conserved among different P2X receptors. Drug Dev Res 61:227-232, 2004.     

Combined effects of sulindac and suberoylanilide hydroxamic acid on apoptosis induction in human lung cancer cells

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. Treatment of cancer cells with HDAC blockers, such as suberoylanilide hydroxamic acid (SAHA), leads to the activation of apoptosis-promoting genes. To enhance proapoptotic efficiency, SAHA has been used in conjunction with radiation, kinase inhibitors, and cytotoxic drugs. In the present study, we show that at the suboptimal dose of 250 muM, sulindac [2-[6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylidene]inden-1-yl]-acetic acid] significantly enhances SAHA-induced growth suppression and apoptosis of A549 human non-small cell lung cancer cells, primarily via enhanced collapse of the mitochondrial membrane potential, release of cytochrome c, and caspase activation. Furthermore, sulindac/SAHA cotreatment induced marked down-regulation of survivin at both the mRNA and protein levels and stimulated the production of reactive oxygen species (ROS), which were blocked by the antioxidant N-acetyl-l-cysteine. Overexpression of survivin was associated with reduced sulindac/SAHA-induced apoptosis of A549 cells, whereas suppression of survivin levels with antisense oligonucleotides or small interfering RNA further sensitized cells to sulindac/SAHA-induced cell death. Our results collectively demonstrate that sulindac/SAHA-induced apoptosis is mediated by ROS-dependent down-regulation of survivin in lung cancer cells.     

3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides as a new class of synthetic histone deacetylase inhibitors. 2. Effect of pyrrole-C2 and/or -C4 substitutions on biological activity

Previous SAR studies (Part 1: Mai, A.; et al. J. Med. Chem. 2003, 46, 512-524) performed on some portions (pyrrole-C4, pyrrole-N1, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1a) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl (1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the present paper, we investigated the effect on anti-HD2 activity of chemical substitutions performed on the pyrrole-C2 ethene chains of 1a-c, which were replaced with methylene, ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl moiety into the 1a chemical structure (IC50(2f) = 0.77 microM; IC50(1a) = 3.8 microM). IC50 values of compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups at the pyrrole-C(4) position a hydrocarbon spacer length ranging from two to five methylenes is well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes) are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives. Particularly, 3a (IC50 = 0.043 microM) showed the same potency as SAHA in inhibiting HD2 in vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was 4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information potentially useful for a future 3D-QSAR study, showed the same (4a,b) or higher (4c,d) HD2 inhibiting activities in comparison with those of the reference drugs. Molecular modeling and docking calculations on the designed compounds performed in parallel with the chemistry work fully supported the synthetic effort and gave insights into the binding mode of the more flexible APHA derivatives (i.e., 3a). Despite the difference of potency between 1b and 3a in the enzyme assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the differentiation assay only at the highest tested dose (48 microM).     

Redox-sensitive iodinated polymersomes carrying histone deacetylase inhibitor as a dual-functional nano-radiosensitizer for enhanced radiotherapy of breast cancer

Radiotherapy (RT) is a frequently used means in clinical tumor treatment. The outcome of RT varies, however, to a great extent, due to RT resistance or intolerable dose, which might be resolved by the development of radio-sensitizing strategies. Here, we report redox-sensitive iodinated polymersomes (RIP) carrying histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA, vorinostat), as a new dual-functional nano-radiosensitizer for breast cancer radiotherapy. SAHA-loaded RIP (RIP-SAHA) with a size of about 101 nm exhibited good colloidal stability while the reduction-activated release of SAHA, giving rise to better antitumor effect to 4T1 breast carcinoma cells than free SAHA. Accordingly, RIP-SAHA combined with a 4 Gy dose of X-ray radiation led to significantly enhanced suppression of 4T1 cells compared with SAHA combined 4 Gy of X-ray radiation, as a result of enhanced DNA damage and impeded DNA damage repair. The pharmacokinetics and biodistribution studies by single-photon emission computed tomography (SPECT) with ¹²⁵I-labeled SAHA (¹²⁵I-SAHA) showed a 17.3-fold longer circulation and 237.7-fold better tumor accumulation of RIP-SAHA over SAHA. The systemic administration of RIP-SAHA greatly sensitized radiotherapy of subcutaneous 4T1 breast tumors and brought about significant inhibition of tumor growth, without causing damages to major organs, compared with radiotherapy alone. RIP not only enhanced SAHA delivery but also acted as a radiosensitizer. RIP-SAHA emerges as a smart dual-functional nano-radiosensitizer to effectively enhance tumor radiotherapy.     

Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.     

Aromatic reduced amide bond peptidomimetics as selective inhibitors of neuronal nitric oxide synthase

Nitric oxide synthase inhibitors could act as important therapies for disorders arising from overstimulation or overexpression of individual nitric oxide synthase (NOS) isoforms. But preservation of physiologically important nitric oxide functions require the use of isoform-selective inhibitors. Recently we reported reduced amide bond pseudodipeptide analogues as potent and selective neuronal nitric oxide synthase (nNOS) inhibitors (Hah, J.-M.; Roman, L. J.; Martasek, P.; Silverman, R. B. J. Med. Chem. 2001, 44, 2667-2670). To increase the lipophilicity a series of aromatic, reduced amide bond analogues (6-25) were designed and synthesized as potential selective nNOS inhibitors. The hypothesized large increase in isoform selectivity of nNOS over inducible NOS was not obtained in this series. However, the high potency with nNOS as well as high selectivity of nNOS over endothelial NOS was retained in some of these compounds (15, 17, 21), as well as good selectivity over inducible NOS. The most potent nNOS inhibitor among these compounds is N-(4S)-[4-amino-5-[2-(2-aminoethyl)phenylamino]-pentyl]-N'-nitroguanidine (17) (K(i) = 50 nM), which also shows the highest selectivity over eNOS (greater than 2100-fold) and 70-fold selectivity over iNOS. Further modification of compound 17 should lead to even more potent and selective nNOS inhibitors.     

Synthesis and pharmacological characterization of nicotinic acetylcholine receptor properties of (+)- and (-)-pyrido-[3,4-b]homotropanes

(+/-)-Pyrido[3,4-b]homotropane [(+/-)-1] is a conformationally rigid analogue of nicotine (2) or nornicotine (3) that showed high affinity for nicotinic acetylcholine receptors. Even though the synthesis and potent activity of this highly interesting compound was originally reported in 1986 (Kanne, D. B.; Ashworth, D. J.; Cheng, M. T.; Mutter, L. C.; Abood, L. G. Synthesis of the first highly potent bridged nicotinoid. 9-Azabicylo[4.2.l]nona[2,3-c]pyridine (pyrido[3,4-b]homotropane). J. Am. Chem. Soc. 1986, 108, 7864-7865), the individual optical isomers have not been prepared and studied. In this study, we report the synthesis of (+)- and (-)-1 and show that (+)-1 has Ki = 1.29 nM at the alpha4beta2* nAChR and has over 260 times higher affinity than (-)-1. Single-crystal X-ray analysis of an intermediate used to prepare the isomers established the absolute stereochemistry as (1S,6S)-(+)-1 and (1R,6R)-(-)-1. Surprisingly, both isomers failed to produce antinociception in the mouse tail-flick and hot-plate assays, engender nicotine-like responding in rat drug discrimination, or alter current amplitude in alpha4beta2- and alpha3beta4-containing cells. However, (-)-1 antagonized nicotine-induced antinociception with an ED50 of 0.07 microg/kg in the tail-flick assay. The reason for this unusual pharmacology is unknown, but it is possible that (-)-1 is acting at a non-epibatidine-sensitive receptor subtype to antagonize nicotine's effects in the tail-flick assay.     

Chronic lead exposure enhances the sympathoexcitatory response associated with P2X4 receptor in rat stellate ganglia

Chronic lead exposure causes peripheral sympathetic nerve stimulation, including increased blood pressure and heart rate. Purinergic receptors are involved in the sympathoexcitatory response induced by myocardial ischemia injury. However, whether P2X4 receptor participates in sympathoexcitatory response induced by chronic lead exposure and the possible mechanisms are still unknown. The aim of this study was to explore the change of the sympathoexcitatory response induced by chronic lead exposure via the P2X4 receptor in the stellate ganglion (SG). Rats were given lead acetate through drinking water freely at doses of 0 g/L (control group), 0.5 g/L (low lead group), and 2 g/L (high lead group) for 1 year. Our results demonstrated that lead exposure caused autonomic nervous dysfunction, including blood pressure and heart rate increased and heart rate variability (HRV) decreased. Western blotting results indicated that after lead exposure, the protein expression levels in the SG of P2X4 receptor, IL‐1β and Cx43 were up‐regulated, the phosphorylation of p38 mitogen‐activated protein kinase (MAPK) was activated. Real‐time PCR results showed that the mRNA expression of P2X4 receptor in the SG was higher in lead exposure group than that in the control group. Double‐labeled immunofluorescence results showed that P2X4 receptor was co‐expressed with glutamine synthetase (GS), the marker of satellite glial cells (SGCs). These changes were positively correlated with the dose of lead exposure. The up‐regulated expression of P2X4 receptor in SGCs of the SG maybe enhance the sympathoexcitatory response induced by chronic lead exposure.     

Potent 4-arylalkyl-substituted 3-isothiazolol GABA(A) competitive/noncompetitive antagonists: synthesis and pharmacology

The GABA(A) agonists muscimol (1), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, gaboxadol, 3), and the partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 6a) and their respective 3-isothiazolol analogues thiomuscimol (2), thio-THIP (4), and thio-4-PIOL (7a) are ligands at the GABA(A) orthosteric (recognition) site. The structure-activity relationships (SARs) between these structures are key elements of a 3D-pharmacophore model for GABA(A) agonists and competitive antagonists [Fr?lund, B.; J?rgensen, A. T.; Tagmose, L.; Stensb?l, T. B.; Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.; Krogsgaard-Larsen, P.; Liljefors, T. J. Med. Chem. 2002, 45, 2454-2468]. Prompted by this model, we now report the synthesis and SAR of a series of analogues of 7a, in which the 4-position of the 3-isothiazolol was substituted by alkyl or bulky aromatic groups such as naphthylmethyl and diphenylalkyl groups (7b-h). The compounds have been pharmacologically characterized using receptor binding assays and two-electrode voltage-clamped Xenopus oocytes expressing alpha1beta3gamma2S- and alpha4beta3delta-containing receptors. The compounds show SARs comparable with those of 6b-h but are generally 5-15 times more potent. The 2-naphthylmethyl, the 1-bromo-2-naphthylmethyl, and the 3,3-diphenylpropyl analogues, compounds 7e, 7f, and 7h, respectively, show affinity in the low-nanomolar range (K(i) 2-10 nM). Interestingly, 7e and 7h exhibited a mixed antagonist profile consisting of a noncompetitive component in the picomolar range and a competitive component at concentrations above 1 nM. This unique profile was shown not to be due to either use dependence or kinetic effects. This antagonist profile of 7e and 7h was particularly pronounced at alpha4beta3delta-containing GABA(A) receptors, which showed three- and 10-fold selectivity for 7h and 6h, respectively.     

2-Aminothiazoles as therapeutic leads for prion diseases

2-Aminothiazoles are a new class of small molecules with antiprion activity in prion-infected neuroblastoma cell lines (J. Virol. 2010, 84, 3408). We report here structure-activity studies undertaken to improve the potency and physiochemical properties of 2-aminothiazoles, with a particular emphasis on achieving and sustaining high drug concentrations in the brain. The results of this effort include the generation of informative structure-activity relationships (SAR) and the identification of lead compounds that are orally absorbed and achieve high brain concentrations in animals. The new aminothiazole analogue (5-methylpyridin-2-yl)-[4-(3-phenylisoxazol-5-yl)-thiazol-2-yl]-amine (27), for example, exhibited an EC(50) of 0.94 μM in prion-infected neuroblastoma cells (ScN2a-cl3) and reached a concentration of ～25 μM in the brains of mice following three days of oral administration in a rodent liquid diet. The studies described herein suggest 2-aminothiazoles as promising new leads in the search for effective therapeutics for prion diseases.     

In vitro plasma stability, permeability and solubility of mercaptoacetamide histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACIs) are emerging as a new class of therapeutic agents with potent antitumor activities in a broad spectrum of human cancers. In this study, the in vitro plasma stability, permeability, solubility, and lipophilicity (logD) of two mercaptoacetamide-based HDACIs (coded as W2 and S2) were evaluated and compared to Vorinostat (SAHA). The results demonstrated that the compounds manifested high solubility in HCl (pH 1.2) but lower in PBS (pH 7.4) than SAHA. Moreover, mercaptoacetamide-based HDACIs exhibited higher lipophilicity values compared to SAHA. The permeability of these compounds was evaluated using the Caco-2 cell monolayer as a model of the intestinal mucosa. The Caco-2 studies revealed that the compounds S2 and W2 are highly permeable with apparent permeability coefficients (P(app)) in the apical to basolateral direction of 7.33x10(-6) and 15.0x10(-6)cm/s, respectively. The in vitro stability was determined in human, mouse, porcine and rat plasma. Data showed that the compound W2 is more stable in human and rat plasma and the S2 is more stable in all plasma species than SAHA. Taken together, these results indicate that the mercaptoacetamide-based HDACIs possess favorable solubility, lipophilicity, permeability and plasma stability features.